Bottom Line:
The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities.In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins.No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

ABSTRACTTwo Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

Fig8: Effect of mutation chlM-2 on gene expression after a shift from dark to light. Cultures of wild type and the chlM-2 mutant grown in the dark at time 0 were shifted into dim light (15 μmol m−2 s−1) and samples for RNA isolation were taken at the time points indicated (hours). The average levels of mRNA accumulation relative to the dark control and corrected for differences in loading ±SEM from at least 3 independent experiments are indicated. CBLP served as a loading control. a Test for light induction of Mg-chelatase genes. b Test for light induction of genes encoding light harvesting chlorophyll a/b binding proteins. c Test for light induction of HSP70 genes

Mentions:
A comparative analysis of changes in expression patterns at the mRNA level induced by a shift of wild-type and mutant cultures from dark to light revealed that light induction of all genes analyzed was maintained in chlM-2 (Fig. 8 and Supplementary Fig. S1) and chlM-1 (Supplementary Fig. S1). The degree and kinetics of mRNA accumulation observed exhibited no significant changes for the majority of genes that are involved in tetrapyrrole biosynthesis (HEMA, ALAD, CRD-1, HEM-15) when chlM-2 and chlM-1 (Supplementary Fig. S1) were compared to wild type. In contrast, the mRNAs for the three subunits of Mg-chelatase (CHLH, CHLD, and CHLI) in the mutants showed a distinctly retarded accumulation when compared to wild type, i.e., 2- to 4-fold lower mRNA levels in the 1 h samples (Fig. 8a and Supplementary Fig. S1). A similar observation was made with the LHC genes tested: The initial time course of mRNA accumulation in the mutants was distinctly attenuated after transition from dark to light when it was compared to wild type, i.e., 1.5- to 2.8-fold lower mRNA levels in the 2 h samples (Fig. 8b and Supplementary Fig. S1). After 4 h of light treatment the degree of accumulation observed is ultimately similar in both mutants and wild type. A delayed mRNA accumulation in the first hours after onset of light was also observed for HSP70A and HSP70B: mRNA accumulation was more than twofold reduced in the 1 h sample in both mutants in comparison to wild type (Fig. 8c and Supplementary Fig. S1). Clearly, in both allelic mutants patterns of RNA accumulation are similar and distinct from those of the wild type control, supporting an influence of defects in CHLM on light induction of the genes tested.Fig. 8

Fig8: Effect of mutation chlM-2 on gene expression after a shift from dark to light. Cultures of wild type and the chlM-2 mutant grown in the dark at time 0 were shifted into dim light (15 μmol m−2 s−1) and samples for RNA isolation were taken at the time points indicated (hours). The average levels of mRNA accumulation relative to the dark control and corrected for differences in loading ±SEM from at least 3 independent experiments are indicated. CBLP served as a loading control. a Test for light induction of Mg-chelatase genes. b Test for light induction of genes encoding light harvesting chlorophyll a/b binding proteins. c Test for light induction of HSP70 genes

Mentions:
A comparative analysis of changes in expression patterns at the mRNA level induced by a shift of wild-type and mutant cultures from dark to light revealed that light induction of all genes analyzed was maintained in chlM-2 (Fig. 8 and Supplementary Fig. S1) and chlM-1 (Supplementary Fig. S1). The degree and kinetics of mRNA accumulation observed exhibited no significant changes for the majority of genes that are involved in tetrapyrrole biosynthesis (HEMA, ALAD, CRD-1, HEM-15) when chlM-2 and chlM-1 (Supplementary Fig. S1) were compared to wild type. In contrast, the mRNAs for the three subunits of Mg-chelatase (CHLH, CHLD, and CHLI) in the mutants showed a distinctly retarded accumulation when compared to wild type, i.e., 2- to 4-fold lower mRNA levels in the 1 h samples (Fig. 8a and Supplementary Fig. S1). A similar observation was made with the LHC genes tested: The initial time course of mRNA accumulation in the mutants was distinctly attenuated after transition from dark to light when it was compared to wild type, i.e., 1.5- to 2.8-fold lower mRNA levels in the 2 h samples (Fig. 8b and Supplementary Fig. S1). After 4 h of light treatment the degree of accumulation observed is ultimately similar in both mutants and wild type. A delayed mRNA accumulation in the first hours after onset of light was also observed for HSP70A and HSP70B: mRNA accumulation was more than twofold reduced in the 1 h sample in both mutants in comparison to wild type (Fig. 8c and Supplementary Fig. S1). Clearly, in both allelic mutants patterns of RNA accumulation are similar and distinct from those of the wild type control, supporting an influence of defects in CHLM on light induction of the genes tested.Fig. 8

Bottom Line:
The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities.In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins.No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

ABSTRACTTwo Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.